Though increasing reports highlight circulating extracellular vesicles (EVs) as potential biomarkers and mediators of neuroinflammation and a reflection of the status of the blood-brain barrier (BBB), their roles in physiology and pathophysiology remain obscure. However, because the blood is exposed to numerous cell types that can contribute EVs, and is subject to hemodynamic, rheological and other factors that dictate these cell?s behaviors, the trigger factors in neuroinflammation that cause EV release and determine their content in vivo still await identification. Without recognition of these trigger factors, the therapeutic prospects for targeting EVs will never be fulfilled. Experiments are therefore proposed to address the current void by using a physiologically relevant Dynamic In Vitro BBB (DIV-BBB) characterized by a 3D hollow cartridge that forms a vessel facsimile, wherein the cell composition and laminar flow of media through the lumen can be controlled to mimic aspects of blood content and circulatory conditions in normal and pathologic states. Specifically, brain microvascular endothelial cells (BMECs) ? the preeminent cellular component of the BBB ? will form the walls of the DIV-BBB, and two variables that are likely salient in dictating the source, number and composition of EVs released at the BBB during neuroinflammation will be evaluated. EVs will be retrieved from the DIV-BBB lumen ? reflecting that population to be found in circulating blood ? and their cell source assessed by a novel fluorescent labeling/FACS separation paradigm, their relative amounts by nanoparticle tracking analysis, and their composition by proteomics. In Aim 1, the effects of flow-induced wall shear stress (WSS) on EV production by BMEC, only, will be investigated, as WSS is a major determinant of BMEC phenotype, and cerebral blood flow ? which directly influences WSS ? is altered in some neuroinflammatory states. WSS will be adjusted to levels typically experienced by capillaries or venules, as WSS declines along the microvascular tree, and absolute WSS value may be a deciding factor in EV release. The DIV-BBB will also be exposed + to TNF-a ? a proinflammatory cytokine prominently increased in blood in many neuroinflammatory conditions ? to resolve whether effects of WSS are influenced by cytokine milieu. In Aim 2, the impact of leukocyte:endothelial interactions on EV production will be examined, as the phenotype of both cell types are significantly altered during the processes of adhesion and transendothelial migration (TEM), and such changes could evoke EV production at the BBB in ways that alter the course of neuroinflammation. Effects of leukocyte:endothelial interactions will be gauged under + flow/WSS and + TNFa, as these variables cooperate in vivo to drive inflammation and, thus, in concert, might engender unique EV populations. Additionally, these interactions and their effects on EVs will further be resolved by using antibodies known to block adhesion or subsequent TEM. Using a simplified, yet sufficiently sophisticated DIV-BBB, this strategy will spotlight ? for the first time ? important variables that separately and/or together are likely to serve as trigger factors for EV release at the BBB during neuroinflammation.